We have shown adenosine 3', 5'-monophosphate (cAMP) and its dibutyryl analog, (But)2cAMP, to promote microtubule dependent, in vitro neurite maturation. In this study N6- monobutyryl cAMP and 8- methylthio-cAMP have been found to stimulate neurite development and derivatives of guanosine 3',5'-monophosphate to inhibit neurite numbers and lengths. At time periods where a pronounced stimulation of neurite maturation was observed in the presence of (But)2cAMP, the incorporation of C14-uridine and H3-amino acids into acid precipitable material was unchanged relative to controls. (But)2cAMP has been shown to promote neurite elongation in the presence of colcemid. Electron micrographs of neurites maintained under these conditions have greater numbers of microtubules per unit area than cultures maintained solely in colcemid supplemented media. A cyto-skeletal function for microtubules is well established, but their role in intracellular movement is less defined. Micro- cinematography of our cultures reveals both colcemid and cytochalasin B- sensitive movements. These studies suggest that derivatives of cAMP enhance neurite maturation via some pathway that stimulates microtubule assembly. Newly formed microtubules appear necessary for the maintenance of neurite integrity and are significant for axoplasmic flow.